LncRNA SNHG10 is downregulated in non-small cell lung cancer and predicts poor survival

LncRNA SNHG10 is downregulated in non-small cell lung cancer and predicts poor survival | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article LncRNA SNHG10 is downregulated in non-small cell lung cancer and predicts poor survival Meng Liang, Linlin Wang, Chuanhua Cao, Shimao Song, feng wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-20439/v4 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Oct, 2020 Read the published version in BMC Pulmonary Medicine → Version 4 posted 2 You are reading this latest preprint version Show more versions Abstract Background : LncRNA SNHG10 has been reported to be an oncogenic lncRNA in liver cancer. However, its roles in non-small cell lung cancer (NSCLC) remains unknown. Methods : Tumor and paired non-tumor tissues were harvested from 62 NSCLC patients. RT-qPCR was used to detect the expression of SNHG10 and miR-21 in tissues. Overexpression experiments were used to evaluate the interaction between SNHG10 and miR-21 in NSCLC cells. CCK-8 assay was used to detect the cell proliferation. Results : We observed the expression of SNHG10 was down-regulated in non-small cell lung cancer (NSCLC) compared with that in non-tumor tissues. Moreover, we found that high expression levels of SNHG10 predicted favorable survival of NSCLC patients, and the expression of miR-21 were increased in NSCLC and inversely correlated with SNHG10 expression. In NSCLC cells, overexpression of SNHG10 resulted in increased miR-21 gene methylation and decreased miR-21 expression. Moreover, overexpression of SNHG10 attenuated the enhancing effect of miR-21 overexpression on cell proliferation. Conclusions : SNHG10 may involve in NSCLC cell proliferation by regulating the miR-21 gene methylation. Pulmonology SNHG10 miR-21 NSCLC methylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Non-small cell lung cancer (NSCLC) is the major subtype of lung cancer and is the main cause of cancer-related deaths worldwide [1]. NSCLC has two major subtypes including lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) [2]. Despite of the advances have been made on the treatment and diagnosis of NSCLC, only less than 15% of NSCLC patients can survive for more than 5 years [3]. Therefore, more effective therapeutic approaches are needed. Smoking is the major risk factor for NSCLC [4]. However, never-smokers also develop NSCLC [5], suggesting the involvement of other factors, such as genetic factors, in the molecular pathogenesis of NSCLC [6]. It has been well established that molecular players participate in nearly all aspects of the occurrence and development of NSCLC [7,8]. Increased understanding of the molecular mechanism of NSCLC provides novel targets for the development of anti-cancer approaches, such as targeted therapy [9,10]. LncRNAs are emerging critical players in cancer biology and they participate in cancer mainly by regulating the expression of cancer-related genes [11,12]. Therefore, lncRNAs are potential targets for cancer targeted therapy [13]. SNHG10 has been characterized as an oncogenic lncRNA in liver cancer [14]. However, we observed the downregulation of SNHG10 in NSCLC and it’s inversely correlation with miR-21 by exploring the TCGA dataset. It is known that miR-21 is a key oncogenic miRNA in cancer [15]. This study was therefore performed to investigate the role of SNHG10 and miR-21in NSCLC. Methods Patients and follow-up This study enrolled a total of 62 NSCLC patients (30 cases of LUAD and 32 cases of LUSC) between May 2013 and January 2015 at Taihe Hospital, and was approved by the Ethics Committee of Taihe hospital. All patients were confirmed by histopathological biopsy, and no patients received any therapy for any clinical disorders within 3 months before this study. Other severe clinical disorders were excluded from these patients. Based on AJCC staging system, there were 28 cases at stage I or II, and 34 cases at stage III or IV. Informed consent was signed by all patients. From the day of admission, the 62 patients were followed up for 5 years. The patients were visited every month through phone call. Patients died of non-NSCLC were excluded from this study. The follow-up was completed by all patients. Tissue collection All patients were subjected to biopsy prior to therapy. During biopsy, NSCLC and paired non-tumor tissues were obtained from each patient. Histopathological exam was used to confirm all of the collected tissues. In addition, tissues were immediately subjected to RNA extraction after collections. Cell culture and transfection To match the patients included in this study, the LUSC cell line KLN 205 and LUAD cell line HCC827 were used. RPMI-1640 medium (90%) and FBS (10%) were used to conduct the cell culture. A 5% CO 2 incubator was used to cultivate both cell lines at 37 °C. SNHG10 expressing vector was constructed using pcDNA3.1 (Invitrogen) as the backbone vector. Mimic of miR-21 and negative control (NC) miRNA were purchased from Sigma-Aldrich. Vectors (1 µg) or miRNAs (40 nM) were transfected into KLN 205 and HCC827 cells (1×10 8 ) using lipofectamine 2000 (Invitrogen). KLN 205 and HCC827 cells (1×10 8 ) were transfected with either NC miRNA or empty vector to serve as NC group. Cells were cultivated for further 48 h prior to the following experiments. RT-qPCR Isolation of RNA from tissues and in vitro cultured cells was performed using Ribozol (Invitrogen). DNase I was used to incubate with RNA samples at 37 °C for 2 h to completely digest genomic DNA. RNA samples were reverse transcribed into cDNA samples using a Reverse Transcription System (A5001, Promega Corporation). With cDNA samples as template, qPCRs were carried out to determine the expression of SNHG10 using SYBR Green Master Mix (Bio-Rad). The internal control of SNHG10 was 18S rRNA. Addition of poly (A) was added to mature miRNAs, following by miRNA reverse transcriptions and miRNA qPCRs to determine the expression of miR-21, and the endogenous control for miR-21 was U6. Three replicates were set for each experiment and Ct values were calculated using the 2 -ΔΔCT method. Methylation-specific PCR (MSP) After transfected with empty vector or SNHG10 expression vector, KLN 205 and HCC827 cells were used to extract genomic DNAs using Genomic DNA Extraction Kit (ab156900, Abcam). DNA samples were converted using DNA Methylation-Gold TM kit (ZYMO RESEARCH). After that, the methylation of miR-21 was evaluated by Taq 2X master mix (NEB). Cell Counting Kit-8 (CCK-8) assay After transfected with empty vector or SNHG10 expression vector, KLN 205 and HCC827 cells were subjected to cell proliferation analysis using CCK-8 kit (Dojindo). Cells were washed with ice-cold PBS, followed by cell counting. After that, 3,000 cells in 0.1 ml medium were transferred to each well of a 96-well plate, followed by cell culture at 37 °C. OD values (450 nm) were measured every 24 h for a total of 4 d. At 4 h before the measurement of OD values, CCK-8 solution was added into each well to reach 10%. Statistical analysis Mean ± SD values were used in this study. Difference between two groups was evaluated by paired t- test. Differences among multiple groups were analyzed by ANOVA (one way) and followed by Tukey’s test. Linear regression was used to evaluate the correlations. The 62 patients were divided into high and low SNHG10 level groups (n = 31, cutoff value was the median expression level of SNHG10 in NSCLC tissues) to analyze survival. K-M method and Log-rank test were used to plot and analyze the survival curves. P < 0.05 was considered as statistically significant. Results Downregulation of SNHG10 is correlated with the poor survival of NSCLC patients In order to investigate the expression of SNHG10 in NSCLC patients, TCGA dataset was used to analyze the expression of SNHG10. The result showed that SNHG10 was downregulated in both LUAD (4.04 vs. 8.20) and LUSC (5.71 vs. 8.29) in comparison to that in tumor tissues. To further confirm the downregulation of SNHG10 in NSCLC, the expression of SNHG10 in NSCLC tissues and its paired non-tumor tissues was evaluated by RT-qPCR. Compared with non-tumor tissues, NSCLC tissues exhibited significantly lower expression levels of SNHG10 (Fig. 1A, p < 0.001). Survival curve analysis revealed that patients in high SNHG10 level group showed higher overall survival rate compared to patients in low SNHG10 level group (Fig. 1B). MiR-21 was upregulated in NSCLC and inversely correlated with SNHG10 RT-qPCR was used to evaluate the miR-21 expression in paired NSCLC and non-tumor tissues. The results showed that miR-21 expression was significantly increased in NSCLC tissues compared with non-tumor tissues (Fig. 2A, p < 0.001). Moreover, the expression levels of miR-21 and SNHG10 were inversely and significantly correlated across NSCLC tissues (Fig. 2B), but not across non-tumor tissues (Fig. 2C) evaluated by correlation analysis. SNHG10 downregulated miR-21 in NSCLC cell through methylation KLN 205 and HCC827 cells were used to overexpress the SNHG10 or miR-21, and RT-qPCR was used to confirm these overexpression (Fig. 3A, p < 0.05). The results showed that SNHG10 overexpression resulted in downregulation of miR-21 expression in KLN 205 and HCC827 cells (Fig. 3B, p 0.05). The effects of SNHG10 overexpression on miR-21 methylation was evaluated by MSP. In addition, SNHG10 overexpression showed significantly increased miR-21 methylation (Fig. 3D). Overexpression of SNHG10 attenuated the enhancing effect of miR-21 overexpression on cell proliferation The roles of SNHG10 and miR-21 in regulating the proliferation of KLN 205 and HCC827 cells were evaluated by CCK-8. Compared with control cells, decreased proliferation of cells was observed after the overexpression of SNHG10, while increased proliferation of cells was observed after the overexpression of miR-21. Moreover, overexpression of SNHG10 attenuated the enhancing effect of miR-21 overexpression on cell proliferation (Fig. 4, p < 0.05). Discussion In the present study, we aimed to investigate the role and underlying mechanism of SNHG10 in NSCLC. Clinical data showed that SNHG10 was downregulated in NSCLC and predicted poor survival of NSCLC patients. Additionally, miR-21 was up-regulated and negatively correlated with SHG10 in NSCLC. In two NSCLC cell lines, we revealed that SNHG10 reduced miR-21 via methylation. Moreover, SNHG10 inhibited the proliferation of NSCLC cells by targeting miR-21. Therefore, SNHG10 is a tumor suppressor in NSCLC. lncRNAs have been found to be involved in the development of cancer. For example, lncRNA DANCR could enhance cancer cell migration and invasion in gastric cancer [16]. LncRNA XIST could induce proliferation in pancreatic cancer cells [17]. A recent study reported that SNHG10 was an oncogenic lncRNA in liver cancer. It is reported that SNHG10 was upregulated in liver cancer and formed a positive feedback loop with its homolog SCARNA13, thereby promoting cancer metastasis [14]. Interestingly, SNHG10 was remarkably downregulated in NSCLC according to our analyses of TCGA dataset. We confirmed this finding by determining the expression of SNHG10 in paired NSCLC and non-tumor tissues. In two NSCLC cell lines, overexpression of SNHG10 resulted in decreased proliferation of NSCLC cells. Therefore, SNHG10 is likely a tumor suppressor lncRNA in NSCLC, and SNHG10 may play different roles in different types of cancer, suggesting that NSCLC and liver cancer may have different molecular pathogenesis. Even with active treatments, such as surgical resection and chemotherapy, the overall survival of NSCLC is still poor [18,19]. In this study, we showed that overexpression of SNHG10 suppressed the proliferation of NSCLC, and high expression levels of SNHG10 were correlated with the favorable survival of NSCLC patients. Therefore, SNHG10 may serve as a target for the treatment of NSCLC. In addition, measuring the expression levels of SNHG10 before therapy may assist the prognosis of NSCLC, thereby guiding the determination of treatments and improve patients’ survival. MiR-21 is a well-characterized oncogenic miRNA that promotes tumorigenesis in many cancers, such as cervical, breast and gastric cancers [20-22]. In NSCLC, miR-21 is a serum biomarker for detection of early-stage NSCLC, and has been found to enhance cancer progression by targeting its targets genes, like SOCS1, PTEN, SOX7 [23-25]. However, the upstream regulators of miR-21 have not been well studied. In this study, we found that miR-21 was negatively correlated with SNHG10 in NSCLC tissues. Moreover, SNHG10 was directly regulated by SNHG10 through methylation, and it was involved in the inhibitory effect of SNHG10 on NSCLC cell proliferation. Hence, SNHG10 is an upstream regulator of miR-21 and can inhibits its oncogenic function in NSCLC. It is worth noting that SNHG10 and miR-21 were only closely correlated across NSCLC tissues, but not across non-tumor tissues. Therefore, certain pathological factors may mediate the interaction between them, and further studies are needed. Conclusion In conclusion, SNHG10 is downregulated, and miR-21 was upregulated in NSCLC. SNHG10 predicts the prognosis of NSCLC, and it can downregulate miR-21 through methylation to suppress the proliferation of cancer cells. Abbreviations NSCLC: non-small cell lung cancer; LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma; MSP: Methylation-specific PCR; CCK-8:Cell Counting Kit-8 assay Declarations Ethics approval and consent to participate The present study was approved by the Ethics Committee of Tianhe Hospital. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. All patients provided written informed consent before their inclusion within the study. Consent for publication All subjects participating in the image acquisition signed the consent form. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests The authors declare that there is no conflict of interests. Funding None Authors' contributions W.F. designed and directed this study. L.M. and W.L.L performed the experiments and wrote the manuscript. C.C.H participated in revision of the manuscript. S.S.M. provided assistance for data analysis. All authors read and approved the manuscript for publication Acknowledgements None References [1] Zappa C, Mousa S A. Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res. 2016, 5(3): 288-300. [2] Rakaee M, Busund L T, Paulsen E E, et al. Prognostic effect of intratumoral neutrophils across histological subtypes of non-small cell lung cancer. Oncotarget. 2016, 7(44): 72184-72196. [3] Aguiar Jr P N, De Mello R A, Hall P, et al. PD-L1 expression as a predictive biomarker in advanced non-small-cell lung cancer: updated survival data. Immunotherapy. 2017, 9(6): 499-506. [4] O’Keeffe L M, Taylor G, Huxley R R, et al. Smoking as a risk factor for lung cancer in women and men: a systematic review and meta-analysis. BMJ Open. 2018, 8(10): e021611. [5] Hung R J, Spitz M R, Houlston R S, et al. Lung Cancer Risk in Never-Smokers of European Descent is Associated With Genetic Variation in the 5p15. 33 TERT-CLPTM1Ll Region. J Thorac Oncol. 2019, 14(8): 1360-1369. [6] Malhotra J, Malvezzi M, Negri E, et al. Risk factors for lung cancer worldwide. Eur Respir J. 2016, 48(3): 889-902. [7] Kadara H, Scheet P, Wistuba I I, et al. Early events in the molecular pathogenesis of lung cancer. Cancer Prev Res (Phila). 2016, 9(7): 518-527. [8] Wood S L, Pernemalm M, Crosbie P A, et al. Molecular histology of lung cancer: from targets to treatments. Cancer Treat Rev. 2015, 41(4): 361-375. [9] Kamphorst A O, Pillai R N, Yang S, et al. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1–targeted therapy in lung cancer patients. Proc Natl Acad Sci U S A. 2017, 114(19): 4993-4998. [10] Barr Kumarakulasinghe N, Zanwijk N, Soo R A. Molecular targeted therapy in the treatment of advanced stage non‐small cell lung cancer (NSCLC). Respirology. 2015, 20(3): 370-378. [11] Schmitt A M, Chang H Y. Long noncoding RNAs in cancer pathways. Cancer Cell. 2016, 29(4): 452-463. [12] Evans J R, Feng F Y, Chinnaiyan A M. The bright side of dark matter: lncRNAs in cancer. J Clin Invest. 2016, 126(8): 2775-2782. [13] Khorkova O, Hsiao J, Wahlestedt C. Basic biology and therapeutic implications of lncRNA. Adv Drug Deliv Rev. 2015, 87: 15-24. [14] Lan T, Yuan K, Yan X, et al. LncRNA SNHG10 facilitates hepatocarcinogenesis and metastasis by modulating its homolog SCARNA13 via a positive feedback loop. Cancer Res. 2019, 79(13): 3220-3234. [15] Pfeffer S R, Yang C H, Pfeffer L M. The role of miR-21 in cancer. Drug Dev Res. 2015, 76(6): 270-277. [16] Mao Z, Li H, Du, B, et al. LncRNA DANCR promotes migration and invasion through suppression of lncRNA-LET in gastric cancer cells. Bioscience reports, 2017, 37(6): BSR20171070. [17] Wei W, Liu Y, Lu Y, et al. LncRNA XIST promotes pancreatic cancer proliferation through miR‐133a/EGFR. Journal of cellular Biochemistry, 2017, 118(10): 3349-3358. [18] Antonia S J, Villegas A, Daniel D, et al. Overall survival with durvalumab after chemoradiotherapy in stage III NSCLC. N Engl J Med. 2018, 379(24): 2342-2350. [19] Alvarez J G B, González-Cao M, Karachaliou N, et al. Advances in immunotherapy for treatment of lung cancer. Cancer Biol Med. 2015, 12(3): 209-222. [20] Park S, Eom K, Kim J, et al. MiR-9, miR-21, and miR-155 as potential biomarkers for HPV positive and negative cervical cancer. BMC cancer, 2017, 17(1): 658. [21] Han J G, Jiang Y D, Zhang C H, et al. A novel panel of serum miR-21/miR-155/miR-365 as a potential diagnostic biomarker for breast cancer. Annals of surgical treatment and research, 2017, 92(2): 55-66. [22] Zheng P, Chen L, Yuan X, et al. Exosomal transfer of tumor-associated macrophage-derived miR-21 confers cisplatin resistance in gastric cancer cells. Journal of Experimental & Clinical Cancer Research, 2017, 36(1): 53. [23] Xue X, Liu Y, Wang Y, et al. MiR-21 and MiR-155 promote non-small cell lung cancer progression by downregulating SOCS1, SOCS6, and PTEN. Oncotarget, 2016, 7(51): 84508. [24] Wang P, Chen D, Ma H, et al. LncRNA MEG3 enhances cisplatin sensitivity in non-small cell lung cancer by regulating miR-21-5p/SOX7 axis. OncoTargets and therapy, 2017, 10: 5137. [25] Zhang H, Mao F, Shen T, et al. Plasma miR‑145, miR‑20a, miR‑21 and miR‑223 as novel biomarkers for screening early‑stage non‑small cell lung cancer. Oncology letters, 2017, 13(2): 669-676. Cite Share Download PDF Status: Published Journal Publication published 20 Oct, 2020 Read the published version in BMC Pulmonary Medicine → Version 4 posted Submission checks completed at journal 01 Sep, 2020 Editorial decision: Accept 01 Sep, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-20439","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":1897137,"identity":"dbe8f456-b95b-4da6-8bfc-4484aef2acae","order_by":0,"name":"Meng Liang","email":"","orcid":"","institution":"Taihe Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Liang","suffix":""},{"id":1897138,"identity":"8d42f52c-c20b-4123-9ee1-ff792073ece9","order_by":1,"name":"Linlin Wang","email":"","orcid":"","institution":"Hubei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Linlin","middleName":"","lastName":"Wang","suffix":""},{"id":1897139,"identity":"88c100b3-d577-4560-9854-b8848fa7f742","order_by":2,"name":"Chuanhua Cao","email":"","orcid":"","institution":"Xiangyang Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chuanhua","middleName":"","lastName":"Cao","suffix":""},{"id":1897140,"identity":"c44db821-02d9-4bbb-8ce6-913fc7f70b87","order_by":3,"name":"Shimao Song","email":"","orcid":"","institution":"Taihe Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shimao","middleName":"","lastName":"Song","suffix":""},{"id":1897141,"identity":"fa6e2fe2-46c2-4e79-a437-9181dfcaf583","order_by":4,"name":"feng wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIie3PMQrCMBTG8YRAXVKypuAh4lSEoldpEZycpeODwusV4kWcExxcSrsKXRQvkO4OOroIr5tDfvP7w/sYi6I/ZBhvQ6gLqRSQE4HcdvtlZh05WaBI8VIYKIlJrjzeUxykYY6H6UBI1rZqje5HmQsQ2elMeezGQZvjKNfgEpGSksGDLpNeGldSE1ehduhmJJ8tzQq6ncysb2hbcnV9PF/1ZqtU48NESb5wmHcfRVEU/fYG1RY53EIfiPUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9773-7193","institution":"Taihe Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"feng","middleName":"","lastName":"wu","suffix":""}],"badges":[],"createdAt":"2020-03-31 11:09:37","currentVersionCode":4,"declarations":"","doi":"10.21203/rs.3.rs-20439/v4","doiUrl":"https://doi.org/10.21203/rs.3.rs-20439/v4","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12890-020-01281-w","type":"published","date":"2020-10-20T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2205153,"identity":"776db0e9-efd5-41e3-83e7-aca50bbc1f25","added_by":"auto","created_at":"2020-09-02 17:00:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":277433,"visible":true,"origin":"","legend":"Downregulation of SNHG10 is correlated with the poor survival of NSCLC patients\nExpression of SNHG10 in paired tissues was determined by RT-qPCR. Levels of SNHG10 expression were compared between NSCLC and non-tumor tissues. Mean values were compared (A). ***, p \u003c 0.001. To analyze survival, the 62 patients were divided into high and low SNHG10 level groups (n = 31, with median expression level of SNHG10 in NSCLC tissues as cutoff value). Survival curves were plotted and compared by log-rank test (B).$\n","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-20439/v4/1.png"},{"id":2205154,"identity":"499b0f79-0f98-4bdd-9d47-e248a309a3c1","added_by":"auto","created_at":"2020-09-02 17:00:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":400266,"visible":true,"origin":"","legend":"MiR-21 was upregulated in NSCLC and inversely correlated with SNHG10\nExpression of miR-21 in paired NSCLC and non-tumor tissues from the 62 patients was determined by RT-qPCR. Levels of miR-21 expression were compared between NSCLC and non-tumor tissues. Mean values were compared (A). ***, p \u003c 0.001. Linear regression was performed to analyze the correlations between SNHG10 and miR-21 across NSCLC tissues (B) and non-tumor tissues (C).\n","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-20439/v4/2.png"},{"id":2205155,"identity":"7ab13d11-6247-4627-a4bf-a07176d30ed2","added_by":"auto","created_at":"2020-09-02 17:00:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":608027,"visible":true,"origin":"","legend":"SNHG10 downregulated miR-21 in NSCLC cell through methylation\nSNHG10 expression vector or miR-21 mimic was transfected into KLN 205 and HCC827 cells. Transfections were confirmed by RT-qPCR (A). The effects of SNHG10 overexpression on miR-21 (B), and the effects of miR-21 overexpression on SNHG10 (C) were also analyzed by RT-qPCR. MSP was performed to analyze the effects of SNHG10 overexpression on miR-21 (D). Mean±SD values were presented and compared. M, methylation; U, un-methylation; *, p \u003c 0.05.\n","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-20439/v4/3.png"},{"id":2205156,"identity":"af236fb9-5bdc-4bc4-9335-1d8fc4148259","added_by":"auto","created_at":"2020-09-02 17:00:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":354660,"visible":true,"origin":"","legend":"SNHG10 overexpression attenuated the enhancing effect of miR-21 overexpression on cell proliferation\nThe roles of SNHG10 and miR-21 in regulating the proliferation of KLN 205 and HCC827 cells were analyzed by CCK-8. Mean ± SD values were presented and compared. *, p \u003c 0.05.\n","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-20439/v4/4.png"},{"id":13587712,"identity":"5ed01864-b135-4f25-b5ed-7b365efe6138","added_by":"auto","created_at":"2021-09-17 04:52:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1119108,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-20439/v4/2c310517-8a3b-4f84-956d-364e56416f0c.pdf"}],"financialInterests":"","formattedTitle":"LncRNA SNHG10 is downregulated in non-small cell lung cancer and predicts poor survival","fulltext":[{"header":"Background","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC) is the major subtype of lung cancer and is the main cause of cancer-related deaths worldwide [1]. NSCLC has two major subtypes including lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) [2]. Despite of the advances have been made on the treatment and diagnosis of NSCLC, only less than 15% of NSCLC patients can survive for more than 5 years [3]. Therefore, more effective therapeutic approaches are needed. Smoking is the major risk factor for NSCLC [4]. However, never-smokers also develop NSCLC [5], suggesting the involvement of other factors, such as genetic factors, in the molecular pathogenesis of NSCLC [6].\u003c/p\u003e\n\u003cp\u003eIt has been well established that molecular players participate in nearly all aspects of the occurrence and development of NSCLC [7,8]. Increased understanding of the molecular mechanism of NSCLC provides novel targets for the development of anti-cancer approaches, such as targeted therapy [9,10]. LncRNAs are emerging critical players in cancer biology and they participate in cancer mainly by regulating the expression of cancer-related genes [11,12]. Therefore, lncRNAs are potential targets for cancer targeted therapy [13]. SNHG10 has been characterized as an oncogenic lncRNA in liver cancer [14]. However, we observed the downregulation of SNHG10 in NSCLC and it\u0026rsquo;s inversely correlation with miR-21 by exploring the TCGA dataset. It is known that miR-21 is a key oncogenic miRNA in cancer [15]. This study was therefore performed to investigate the role of SNHG10 and miR-21in NSCLC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and follow-up\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study enrolled a total of 62 NSCLC patients (30 cases of LUAD and 32 cases of LUSC) between May 2013 and January 2015 at Taihe Hospital, and was approved by the Ethics Committee of Taihe hospital. All patients were confirmed by histopathological biopsy, and no patients received any therapy for any clinical disorders within 3 months before this study. Other severe clinical disorders were excluded from these patients. Based on AJCC staging system, there were 28 cases at stage I or II, and 34 cases at stage III or IV. Informed consent was signed by all patients. From the day of admission, the 62 patients were followed up for 5 years. The patients were visited every month through phone call. Patients died of non-NSCLC were excluded from this study. The follow-up was completed by all patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients were subjected to biopsy prior to therapy. During biopsy, NSCLC and paired non-tumor tissues were obtained from each patient. Histopathological exam was used to confirm all of the collected tissues. In addition, tissues were immediately subjected to RNA extraction after collections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo match the patients included in this study, the LUSC cell line KLN 205 and LUAD cell line HCC827 were used. RPMI-1640 medium (90%) and FBS (10%) were used to conduct the cell culture. A 5% CO\u003csub\u003e2\u003c/sub\u003e incubator was used to cultivate both cell lines at 37 \u0026deg;C. SNHG10 expressing vector was constructed using pcDNA3.1 (Invitrogen) as the backbone vector. Mimic of miR-21 and negative control (NC) miRNA were purchased from Sigma-Aldrich. Vectors (1 \u0026micro;g) or miRNAs (40 nM) were transfected into KLN 205 and HCC827 cells (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e) using lipofectamine 2000 (Invitrogen). KLN 205 and HCC827 cells (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e) were transfected with either NC miRNA or empty vector to serve as NC group. Cells were cultivated for further 48 h prior to the following experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation of RNA from tissues and \u003cem\u003ein vitro\u003c/em\u003e cultured cells was performed using Ribozol (Invitrogen). DNase I was used to incubate with RNA samples at 37 \u0026deg;C for 2 h to completely digest genomic DNA. RNA samples were reverse transcribed into cDNA samples using a Reverse Transcription System (A5001, Promega Corporation). With cDNA samples as template, qPCRs were carried out to determine the expression of SNHG10 using SYBR Green Master Mix (Bio-Rad). The internal control of SNHG10 was 18S rRNA. Addition of poly (A) was added to mature miRNAs, following by miRNA reverse transcriptions and miRNA qPCRs to determine the expression of miR-21, and the endogenous control for miR-21 was U6. Three replicates were set for each experiment and Ct values were calculated using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethylation-specific PCR (MSP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfected with empty vector or SNHG10 expression vector, KLN 205 and HCC827 cells were used to extract genomic DNAs using Genomic DNA Extraction Kit (ab156900, Abcam). DNA samples were converted using DNA Methylation-Gold\u003csup\u003eTM\u003c/sup\u003e kit (ZYMO RESEARCH). After that, the methylation of miR-21 was evaluated by Taq 2X master mix (NEB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Counting Kit-8 (CCK-8) assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfected with empty vector or SNHG10 expression vector, KLN 205 and HCC827 cells were subjected to cell proliferation analysis using CCK-8 kit (Dojindo). Cells were washed with ice-cold PBS, followed by cell counting. After that, 3,000 cells in 0.1 ml medium were transferred to each well of a 96-well plate, followed by cell culture at 37 \u0026deg;C. OD values (450 nm) were measured every 24 h for a total of 4 d. At 4 h before the measurement of OD values, CCK-8 solution was added into each well to reach 10%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean \u0026plusmn; SD values were used in this study. Difference between two groups was evaluated by paired \u003cem\u003et-\u003c/em\u003etest. Differences among multiple groups were analyzed by ANOVA (one way) and followed by Tukey\u0026rsquo;s test. Linear regression was used to evaluate the correlations. The 62 patients were divided into high and low SNHG10 level groups (n = 31, cutoff value was the median expression level of SNHG10 in NSCLC tissues) to analyze survival. K-M method and Log-rank test were used to plot and analyze the survival curves. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDownregulation of \u003c/strong\u003e\u003cstrong\u003eSNHG10 is correlated with the poor survival of NSCLC patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to investigate the expression of SNHG10 in NSCLC patients, TCGA dataset was used to analyze the expression of SNHG10. The result showed that SNHG10 was downregulated in both LUAD (4.04 vs. 8.20) and LUSC (5.71 vs. 8.29) in comparison to that in tumor tissues. To further confirm the downregulation of SNHG10 in NSCLC, the expression of SNHG10 in NSCLC tissues and its paired non-tumor tissues was evaluated by RT-qPCR. Compared with non-tumor tissues, NSCLC tissues exhibited significantly lower expression levels of SNHG10 (Fig. 1A, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Survival curve analysis revealed that patients in high SNHG10 level group showed higher overall survival rate compared to patients in low SNHG10 level group (Fig. 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-21 was upregulated in NSCLC and inversely correlated with SNHG10\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR was used to evaluate the miR-21 expression in paired NSCLC and non-tumor tissues. The results showed that miR-21 expression was significantly increased in NSCLC tissues compared with non-tumor tissues (Fig. 2A, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Moreover, the expression levels of miR-21 and SNHG10 were inversely and significantly correlated across NSCLC tissues (Fig. 2B), but not across non-tumor tissues (Fig. 2C) evaluated by correlation analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSNHG10 downregulated miR-21 in NSCLC cell through methylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKLN 205 and HCC827 cells were used to overexpress the SNHG10 or miR-21, and RT-qPCR was used to confirm these overexpression (Fig. 3A, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The results showed that SNHG10 overexpression resulted in downregulation of miR-21 expression in KLN 205 and HCC827 cells (Fig. 3B, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), while miR-21 overexpression did not affect the SNHG10 expression in KLN 205 and HCC827 cells (Fig. 3C, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). The effects of SNHG10 overexpression on miR-21 methylation was evaluated by MSP. In addition, SNHG10 overexpression showed significantly increased miR-21 methylation (Fig. 3D).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of SNHG10 attenuated the enhancing effect of miR-21 overexpression on cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe roles of SNHG10 and miR-21 in regulating the proliferation of KLN 205 and HCC827 cells were evaluated by CCK-8. Compared with control cells, decreased proliferation of cells was observed after the overexpression of SNHG10, while increased proliferation of cells was observed after the overexpression of miR-21. Moreover, overexpression of SNHG10 attenuated the enhancing effect of miR-21 overexpression on cell proliferation (Fig. 4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we aimed to investigate the role and underlying mechanism of SNHG10 in NSCLC. Clinical data showed that SNHG10 was downregulated in NSCLC and predicted poor survival of NSCLC patients. Additionally, miR-21 was up-regulated and negatively correlated with SHG10 in NSCLC. In two NSCLC cell lines, we revealed that SNHG10 reduced miR-21 via methylation. Moreover, SNHG10 inhibited the proliferation of NSCLC cells by targeting miR-21. Therefore, SNHG10 is a tumor suppressor in NSCLC.\u003c/p\u003e\n\u003cp\u003elncRNAs have been found to be involved in the development of cancer. For example, lncRNA DANCR could enhance cancer cell migration and invasion in gastric cancer [16]. LncRNA XIST could induce proliferation in pancreatic cancer cells [17]. A recent study reported that SNHG10 was an oncogenic lncRNA in liver cancer. It is reported that SNHG10 was upregulated in liver cancer and formed a positive feedback loop with its homolog SCARNA13, thereby promoting cancer metastasis [14]. Interestingly, SNHG10 was remarkably downregulated in NSCLC according to our analyses of TCGA dataset. We confirmed this finding by determining the expression of SNHG10 in paired NSCLC and non-tumor tissues. In two NSCLC cell lines, overexpression of SNHG10 resulted in decreased proliferation of NSCLC cells. Therefore, SNHG10 is likely a tumor suppressor lncRNA in NSCLC, and SNHG10 may play different roles in different types of cancer, suggesting that NSCLC and liver cancer may have different molecular pathogenesis.\u003c/p\u003e\n\u003cp\u003eEven with active treatments, such as surgical resection and chemotherapy, the overall survival of NSCLC is still poor [18,19]. In this study, we showed that overexpression of SNHG10 suppressed the proliferation of NSCLC, and high expression levels of SNHG10 were correlated with the favorable survival of NSCLC patients. Therefore, SNHG10 may serve as a target for the treatment of NSCLC. In addition, measuring the expression levels of SNHG10 before therapy may assist the prognosis of NSCLC, thereby guiding the determination of treatments and improve patients\u0026rsquo; survival.\u003c/p\u003e\n\u003cp\u003eMiR-21 is a well-characterized oncogenic miRNA that promotes tumorigenesis in many cancers, such as cervical, breast and gastric cancers [20-22]. In NSCLC, miR-21 is a serum biomarker for detection of early-stage NSCLC, and has been found to enhance cancer progression by targeting its targets genes, like SOCS1, PTEN, SOX7 [23-25]. However, the upstream regulators of miR-21 have not been well studied. In this study, we found that miR-21 was negatively correlated with SNHG10 in NSCLC tissues. Moreover, SNHG10 was directly regulated by SNHG10 through methylation, and it was involved in the inhibitory effect of SNHG10 on NSCLC cell proliferation. Hence, SNHG10 is an upstream regulator of miR-21 and can inhibits its oncogenic function in NSCLC. It is worth noting that SNHG10 and miR-21 were only closely correlated across NSCLC tissues, but not across non-tumor tissues. Therefore, certain pathological factors may mediate the interaction between them, and further studies are needed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, SNHG10 is downregulated, and miR-21 was upregulated in NSCLC. SNHG10 predicts the prognosis of NSCLC, and it can downregulate miR-21 through methylation to suppress the proliferation of cancer cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNSCLC: non-small cell lung cancer; LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma; MSP: Methylation-specific PCR; CCK-8:Cell Counting Kit-8 assay\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Ethics Committee of Tianhe Hospital. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. All patients provided written informed consent before their inclusion within the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll subjects participating in the image acquisition signed the consent form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.F. designed and directed this study. L.M. and W.L.L performed the experiments and wrote the manuscript. C.C.H participated in revision of the manuscript. S.S.M. provided assistance for data analysis. All authors read and approved the manuscript for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e[1] Zappa C, Mousa S A. Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res. 2016, 5(3): 288-300.\u003c/p\u003e\n\u003cp\u003e[2] Rakaee M, Busund L T, Paulsen E E, et al. Prognostic effect of intratumoral neutrophils across histological subtypes of non-small cell lung cancer. Oncotarget. 2016, 7(44): 72184-72196.\u003c/p\u003e\n\u003cp\u003e[3] Aguiar Jr P N, De Mello R A, Hall P, et al. PD-L1 expression as a predictive biomarker in advanced non-small-cell lung cancer: updated survival data. 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Cancer Treat Rev. 2015, 41(4): 361-375.\u003c/p\u003e\n\u003cp\u003e[9] Kamphorst A O, Pillai R N, Yang S, et al. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1\u0026ndash;targeted therapy in lung cancer patients. Proc Natl Acad Sci U S A. 2017, 114(19): 4993-4998.\u003c/p\u003e\n\u003cp\u003e[10] Barr Kumarakulasinghe N, Zanwijk N, Soo R A. Molecular targeted therapy in the treatment of advanced stage non‐small cell lung cancer (NSCLC). Respirology. 2015, 20(3): 370-378.\u003c/p\u003e\n\u003cp\u003e[11] Schmitt A M, Chang H Y. Long noncoding RNAs in cancer pathways. Cancer Cell. 2016, 29(4): 452-463.\u003c/p\u003e\n\u003cp\u003e[12] Evans J R, Feng F Y, Chinnaiyan A M. The bright side of dark matter: lncRNAs in cancer. J Clin Invest. 2016, 126(8): 2775-2782.\u003c/p\u003e\n\u003cp\u003e[13] Khorkova O, Hsiao J, Wahlestedt C. Basic biology and therapeutic implications of lncRNA. Adv Drug Deliv Rev. 2015, 87: 15-24.\u003c/p\u003e\n\u003cp\u003e[14] Lan T, Yuan K, Yan X, et al. LncRNA SNHG10 facilitates hepatocarcinogenesis and metastasis by modulating its homolog SCARNA13 via a positive feedback loop. Cancer Res. 2019, 79(13): 3220-3234.\u003c/p\u003e\n\u003cp\u003e[15] Pfeffer S R, Yang C H, Pfeffer L M. The role of miR-21 in cancer. Drug Dev Res. 2015, 76(6): 270-277.\u003c/p\u003e\n\u003cp\u003e[16] Mao Z, Li H, Du, B, et al. LncRNA DANCR promotes migration and invasion through suppression of lncRNA-LET in gastric cancer cells. Bioscience reports, 2017, 37(6): BSR20171070.\u003c/p\u003e\n\u003cp\u003e[17] Wei W, Liu Y, Lu Y, et al. LncRNA XIST promotes pancreatic cancer proliferation through miR‐133a/EGFR. Journal of cellular Biochemistry, 2017, 118(10): 3349-3358.\u003c/p\u003e\n\u003cp\u003e[18] Antonia S J, Villegas A, Daniel D, et al. Overall survival with durvalumab after chemoradiotherapy in stage III NSCLC. N Engl J Med. 2018, 379(24): 2342-2350.\u003c/p\u003e\n\u003cp\u003e[19] Alvarez J G B, Gonz\u0026aacute;lez-Cao M, Karachaliou N, et al. Advances in immunotherapy for treatment of lung cancer. Cancer Biol Med. 2015, 12(3): 209-222.\u003c/p\u003e\n\u003cp\u003e[20] Park S, Eom K, Kim J, et al. MiR-9, miR-21, and miR-155 as potential biomarkers for HPV positive and negative cervical cancer. BMC cancer, 2017, 17(1): 658.\u003c/p\u003e\n\u003cp\u003e[21] Han J G, Jiang Y D, Zhang C H, et al. A novel panel of serum miR-21/miR-155/miR-365 as a potential diagnostic biomarker for breast cancer. Annals of surgical treatment and research, 2017, 92(2): 55-66.\u003c/p\u003e\n\u003cp\u003e[22] Zheng P, Chen L, Yuan X, et al. Exosomal transfer of tumor-associated macrophage-derived miR-21 confers cisplatin resistance in gastric cancer cells. Journal of Experimental \u0026amp; Clinical Cancer Research, 2017, 36(1): 53.\u003c/p\u003e\n\u003cp\u003e[23] Xue X, Liu Y, Wang Y, et al. MiR-21 and MiR-155 promote non-small cell lung cancer progression by downregulating SOCS1, SOCS6, and PTEN. Oncotarget, 2016, 7(51): 84508.\u003c/p\u003e\n\u003cp\u003e[24] Wang P, Chen D, Ma H, et al. LncRNA MEG3 enhances cisplatin sensitivity in non-small cell lung cancer by regulating miR-21-5p/SOX7 axis. OncoTargets and therapy, 2017, 10: 5137.\u003c/p\u003e\n\u003cp\u003e[25] Zhang H, Mao F, Shen T, et al. Plasma miR‑145, miR‑20a, miR‑21 and miR‑223 as novel biomarkers for screening early‑stage non‑small cell lung cancer. Oncology letters, 2017, 13(2): 669-676.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SNHG10, miR-21, NSCLC, methylation","lastPublishedDoi":"10.21203/rs.3.rs-20439/v4","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-20439/v4","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: LncRNA SNHG10 has been reported to be an oncogenic lncRNA in liver cancer. However, its roles in non-small cell lung cancer (NSCLC) remains unknown. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Tumor and paired non-tumor tissues were harvested from 62 NSCLC patients. RT-qPCR was used to detect the expression of SNHG10 and miR-21 in tissues. Overexpression experiments were used to evaluate the interaction between SNHG10 and miR-21 in NSCLC cells. CCK-8 assay was used to detect the cell proliferation. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: We observed the expression of SNHG10 was down-regulated in non-small cell lung cancer (NSCLC) compared with that in non-tumor tissues. Moreover, we found that high expression levels of SNHG10 predicted favorable survival of NSCLC patients, and the expression of miR-21 were increased in NSCLC and inversely correlated with SNHG10 expression. In NSCLC cells, overexpression of SNHG10 resulted in increased miR-21 gene methylation and decreased miR-21 expression. Moreover, overexpression of SNHG10 attenuated the enhancing effect of miR-21 overexpression on cell proliferation. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: SNHG10 may involve in NSCLC cell proliferation by regulating the miR-21 gene methylation.\u003c/p\u003e","manuscriptTitle":"LncRNA SNHG10 is downregulated in non-small cell lung cancer and predicts poor survival","msid":"","msnumber":"","nonDraftVersions":[{"code":4,"date":"2020-09-02 16:57:28","doi":"10.21203/rs.3.rs-20439/v4","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2020-09-01T12:00:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept","date":"2020-09-01T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":3,"date":"2020-08-20 17:56:45","doi":"10.21203/rs.3.rs-20439/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2020-08-18T12:00:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2020-08-18T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-07-29 15:36:40","doi":"10.21203/rs.3.rs-20439/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-07-24T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-07-22T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-07-21T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-07-21T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-04-03 14:52:41","doi":"10.21203/rs.3.rs-20439/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-05-19T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-05-18T12:00:00+00:00","index":5,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-05-17T12:00:00+00:00","index":4,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-05-17T12:00:00+00:00","index":2,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nThe authors have submitted a MS aimed at demonstrating SNHG10 down-regulation and mir21 up-regulation in NSCLC\nSNHG10 is a prognostic factor and down.regulates miR-21 via methylation leading to reduced cells proliferation\nSurvival curves are consistent with these results\nThe MS is clear and straighforward and the conclusion are consistent however:\n\na) Discussion is too short and the results need much more insight\nb) Fig 4a and -b look the very same\n\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **No**\n* Are the conclusions drawn adequately supported by the data shown?: **Yes**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I am able to assess the statistics**\n* Quality of written English: **Needs some language corrections before being published**\n* Declaration of competing interests: **I declare that i have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please publish my name with my report.**\n"},{"type":"reviewerAgreed","content":"","date":"2020-04-16T12:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-04-15T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-04-08T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-04-08T12:00:00+00:00","index":1,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nWe are facing a paper referring the results of a research discovering marked survival advantage over a five period time in 62 patients divided by the overexpression of an oncogene in lung cancer and its inverse correlation with a miRNA. Therapeutical implications are suggested from this discover.\nFurther, major evaluation are needed about statistic power of this little population of patients and major edition to english language.* Are the methods appropriate and well described?: **Unable to assess**\n* Does the work include the necessary controls?: **Unable to assess**\n* Are the conclusions drawn adequately supported by the data shown?: **Unable to assess**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I recommend additional statistical review**\n* Quality of written English: **Not suitable for publication unless extensively edited**\n* Declaration of competing interests: **We are facing a paper referring the results of a research discovering marked survival advantage over a five period time in 62 patients divided by the overexpression of an oncogene in lung cancer and its inverse correlation with a miRNA. Therapeutical implications are suggested from this discover.\nFurther, major evaluation are needed about statistic power of this little population of patients and major edition to english language.**\n* I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published.: **\nI agree to the open peer review policy of the journal**\n"},{"type":"reviewersInvited","content":"","date":"2020-04-07T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-03-31T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-03-30T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-03-30T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-03-30T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6d0be875-8e2a-460d-8126-c1b4d3d1c9bf","owner":[],"postedDate":"September 2nd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":334835,"name":"Pulmonology"}],"tags":[],"updatedAt":"2020-10-25T15:02:42+00:00","versionOfRecord":{"articleIdentity":"rs-20439","link":"https://doi.org/10.1186/s12890-020-01281-w","journal":{"identity":"bmc-pulmonary-medicine","isVorOnly":false,"title":"BMC Pulmonary Medicine"},"publishedOn":"2020-10-20 12:00:00","publishedOnDateReadable":"October 20th, 2020"},"versionCreatedAt":"2020-09-02 16:57:28","video":"","vorDoi":"10.1186/s12890-020-01281-w","vorDoiUrl":"https://doi.org/10.1186/s12890-020-01281-w","workflowStages":[]},"version":"v4","identity":"rs-20439","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-20439","identity":"rs-20439","version":["v4"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}